Eukaryotic cells are characterized by organelles, sites of biochemical specialization. Nucleus/cytosol exchange is essential for all eukaryotic organisms as RNAs are synthesized in the nucleus and are transported to the cytosol where they function in protein synthesis and many proteins synthesized in the cytosol function in the nucleus in processes such as mitosis, DNA replication, and RNA synthesis. The powerful yeast genetic system will be employed to study the mechanism(s) controlling nucleus/cytosol exchange and how exchange is coupled to RNA processing. Aim 1. To test the hypothesis that Rna1p participates directly in mRNA nuclear export. The Ran-GTPase cycle is required for nuclear import and export and RNA processing, but the cellular distribution of Rnalp, a regulatory protein of this cycle, raises questions as to whether it has a direct role in export. Aim 2. To identify gene products that function in the Ran-independent pathway for nuclear export of mRNAs encoding stress proteins (Hsp). Previous studies showed that Hsp mRNAs exit the nucleus via a Ran-independent path. In situ hybridization will be used to identify mutants defective in nuclear export of Hsp mRNAs. Aim 3. To test the hypothesis that tRNA nuclear export is Ran-independent in yeast and to identify the gene products that function in tRNA nuclear export. Others have shown that in mammalian cells tRNAs exit the nucleus via a Ran-independent pathway. In situ hybridization and the 3-hybrid technique will be used to study the nucleus/cytosol distribution of yeast precursor and mature tRNAs and to identify mutants with altered distribution. Aim 4. To distinguish between roles of Reg1p in regulating the Ran-GTPase cycle components or a Ran-independent path of nucleus/cytosol exchange. Reg1 is a suppressor of an RNA1 allele. To determine whether Reg1p modulates the activities of the Ran-GTPase cycle components, their subcellular locations or, instead, regulates Ran-independent pathways, a combination of genetics, immunofluorescence, in situ hybridization and biochemical assays will be employed. Aim5. To test the hypothesis that the Ran-GTPase pathway functions in processes other than nucleus/cytosol exchange. The Ran pathway has been implicated in a variety of cellular processes in addition to nucleus/cytosol exchange. The PI's lab generated an allele of the gene encoding the Ran GAP that appears not to affect nucleus/cytosol exchange, but rather affects cell cycle progression. To test the model that this GAP participates in other cellular processes, second-site and multicopy suppressors of this mutant allele will be studied and the proteins that interact with Rna1p will be identified by use of the 2-hybrid technique.